investigations of the biology of peranema trichophorum ...anterior end and a tapering posterior end...

279

Investigations of the Biology of Peranema trichophorum(Euglenineae)

By Y. T. CHEN(University of Pekifig; from the Botany School, Cambridge, England)

SUMMARY

The feeding apparatus of Peranema trichophorum, consisting of cytostome and rod-organ, is independent of the reservoir system; the latter is the same in structure andfunction as that of other Euglenineae. There are two flagella, one directed forward,the other backward and adherent to the ventral body surface. The anterior flagellumis longer and thicker than the adherent one. Both flagella are composed of a centralcore and an outer sheath. Electron micrographs suggest that the core consists of manylongitudinal fibrils, and the sheath of many short fibrils radiating from the core, givingthe whole flagellum the appearance of a test-tube brush. Treatment with certain protein-dispersing agents cause the unfixed anterior flagellum to dissociate into three fibrils.

Peranema multiplies freely on a diet of living yeast-cells; dead yeast is not suitable.Euglena viridis, E. gracilis, and certain other unicellular algae can also serve as food.Egg-yolk, and especially milk, can be used to maintain bacteria-free pure cultures.Casein is suitable in combination with soil-extract or beef-extract, but never as goodas milk. With the latter the individuals are larger and more numerous than with yeastas food, although the cultures decline earlier. Clear liquid media of many variouskinds did not support growth: particulate food seems to be essential.

Peranema is capable of ingesting a great variety of living organisms provided theseare motionless. Small organisms are swallowed whole; larger ones are either engulfedor cut open by the rod-organ and their contents sucked out. The rod-organ can beprotruded out of the cytostome and used in holding on to, and cutting, the periplastof the prey. Starch-grains, oil-droplets, and protein-particles are engulfed and digested.The main food reserves are paramylon-granules and oil-droplets.

H+ ions, decomposition products of proteins, and other substances diffusing out ofliving, and particularly dead, organisms attract gliding Peranema. Chemotaxis playsan important role in leading Peranema to its prey.

Both gliding and swimming have been observed in normal individuals, althoughthe latter is less frequent. While there can be no doubt that swimming results from theaction of the anterior flagellum, it does not seem to play an appreciable part in gliding.Nothing is known about the function of the adherent flagellum.

CONTENTSP A G E

I N T R O D U C T I O N . . . . . . . . . . . . . 2 8 0M O R P H O L O G Y . . . . . . . . . . . . . 2 8 1C U L T I V A T I O N A N D G R O W T H . . . . . . . . . . 4 8 5

1 . P r e l i m i n a r y E x p e r i m e n t s . . . . . . . . . . 2 8 5z . B a c t e r i a - f r e e C u l t u r e s . . . . . . . . . . 2 8 8

F E E D I N G H A B I T S . . . . . . . . . . . . 2 9 0D I G E S T I O N • 2 9 6M O V E M E N T A N D C H E M O T A X I S 3 0 0

M o v e m e n t . . . . . . . . . . . . . 3 0 0C h e m o t a x i s . . . . . . . . . . . . 3 0 3

R E F E R E N C E S 3 0 6

[Quarterly Journal of Microscopical Science, Vol. 91, part 3, September 1950.]

280 Chen—Biology of Peranema trichophorum

BINTRODUCTION

ERANEMA trichophorum (Ehrbg.) Stein, a colourless member of theEuglenineae, is one of the largest zootrophic flagellates and often used

for demonstration on account of its conspicuous flagellum. It was, therefore,deemed useful to find reliable methods of maintaining it in the laboratory,since previous attempts at cultivation appeared to rely too much on chance orgave rise to too complex conditions. It was hoped, moreover, that Peranemawould be found suitable for studying the zootrophic habit of nutrition, ofwhich not much is known in flagellates. Bacteria-free cultures of Peranemahave not previously been obtained, and the nutritional requirements of theorganism were practically unknown.

The structure and function of the rod-organ and the reservoir system havelong been matters of controversy. Most of the earlier writers (Stein, 1878;Butschli, 1876, 1883-7; Saville Kent, 1880-2) believed that the rod-organfunctions as a buccal tube through which food passes. This was denied byKlebs (1883, 1893) who accurately described its mode of action and recognizedits function in cutting and piercing the pellicle of the prey.

While the structure of the food-ingesting apparatus of Peranema was notunderstood by earlier writers, two different opinions have appeared in morerecent publications. It is widely believed that the reservoir system, althoughof much the same structure as in phototrophic Euglenineae, is used in thisorganism for ingesting food particles, and that the rod-organ, which is attachedto the reservoir, functions mainly as a mechanical support (Jollos, 1925;Hall and Powell, 1927, 1928; Hall, 1933; Lackey, 1933; see also Schaeffer,1918, for Jenningsia, and Loefer, 1931, for Heteronema). The other opinionis that, in addition to the reservoir system (which has nothing to do withingestion), there is another opening, connected with the rod-organ, and usedfor feeding (Brown, 1930, Pitelka, 1945; see also Rhodes, 1926, for Hetero-nema). Hyman (1936), although accepting the conception that the reservoiris used in feeding, confusedly described an independent rod-organ used in'strengthening the food-ingesting passage'. It seemed necessary to observethe feeding habits of living individuals in order to clarify the position.

The movements of Peranema have been studied by Mast (1912), Lowndes(1936, 1941, 1944, &c.), and others. Lowndes, making use of high-speedcinematography, confirmed Gray's suggestion (1931) that the flagellum is anactive unit and is capable of generating at least a part of its own energy. Therelation between body movement and flagellar activity, the function of theflagellum, and the chemotaxis of this organism have not hitherto receivedattention.

Peranema was collected from stagnant waters containing plant residues.Thriving impure and pure cultures in various media have been obtained.With this material the morphology, growth, feeding habits, movements, andchemotaxis have been investigated in the hope of presenting a more completepicture of a common but surprisingly little-known species and contributing

Quart. Journ. Micr. Set. Third Series, Vol. gi

iA. F. W. HUGHES—PLATE I

Quart. Journ. Micr. Sci. Third Series, Vol. 91

A. F. W. HUGHES—PLATE II

Quart. jfourn. Micr. Set. Third Series, Vol. 91

A. F. W. HUGHES—PLATE III

Quart. Journ. Micr. Sci. Third Series, Vol. gi

A. F. W. HUGHES—PLATE IV

Chen—Biology of Peranetna trichophorum 281

to a better understanding of the relationship between coloured and colourlessEuglenineae.

MORPHOLOGY

The body of Peranetna trichophorum is irregularly sack-shaped, with itsanterior end tapering and its posterior end rounded, truncated, or sometimes(especially when the organism is poorly nourished) prolonged on one side

into a pointed, tail-like process mistaken by James-Clark (1868) for a rudi-mentary trailing flagellum and used by Skuja (1948) as a specific character.

The dimensions of the body vary with the environmental conditions andhave been differently given by previous workers. Newly collected individualsare usually small, with an average size of 42 X 16/u. Under favourable culturalconditions, e.g. in cultures with milk as food, the size increases to 65 X 25/x.on the average, and the individuals are visible to the naked eye as small whitespecks. In cultures with the yeast Saccharomyces exiguus as food the averagesize is 52 X 17 fj..

The periplast is finely striated (Text-fig. 1, A, B). The striations are onlyslightly oblique. Cross-striations have not been seen. Iodine treatment, silverimpregnation, night-blue staining, and dark ground illumination render the


striae more visible. Running along the striae there are many elliptical or rod-shaped granules, visible in living cells under dark ground illumination orafter vital staining (Klebs, 1883; chondriome of Hall, 1926; protrichocystsof Chadefaud, 1938; corps muciferes of Hollande, 1942). In silver-impregnatedpreparations the striations are seen to converge near the posterior end of thebody as they approach a slit-like unimpregnated region, probably the areaof defaecation (Hollande, 1942).

Scattered in the cytoplasm are many paramylon granules. They are disk-shaped with a thick rim. When observed from the side they are ovoid. Thenumber and size of these granules vary considerably: they are fewer andsmaller (with a diameter of about 1-5/*) in individuals fed with S. exiguus,more numerous and larger (diameter about 3 fi) in individuals fed with starch,casein, or milk.

The reservoir, situated in the anterior quarter of the body, is connectedby a canal with an opening lying slightly to the right of the anterior end. Itdivides the latter, as seen in longitudinal optical sections, into a larger and asmaller lip (Text-fig. 1). Posterior to the reservoir is a simple contractilevacuole, the pulsation of which can easily be observed.

The rod-organ is adjacent to the reservoir. It is composed of two parallelrods each of which is hook-shaped with a thickened and slightly curvedanterior end and a tapering posterior end (Text-fig. 1). According to Chadefaud(1938) the rods are semicircular in cross-section, i.e. with a furrow on theinner side. They have been mistaken by many workers for the wall of a canal,called the siphon. In agreement with Brown (1930) and Hyman (1936) it wasfound that the rod-organ is not connected with any part of the reservoirsystem. The anterior end of the rod-organ is attached to a hyaline, roughlysemicircular zone, the position of which is lateral and ventral to, but notconnected with, the reservoir canal (Text-fig, IB). As this zone usually over-laps the reservoir canal, the attached rod-organ also appears to be connectedwith the reservoir. However, normally gliding individuals have repeatedlybeen observed with the hyaline zone and the rod-organ completely separatedfrom any part of the reservoir system (Text-fig. 1, B, C). A lateral view infixed and stained specimens shows the rod-organ again to be ventral to, butindependent of, the reservoir. The clear zone in front of the rod-organ isregarded by Brown (1930) and Pitelka (1945), but not by most other authors,as the true ingestive opening. Hyman (1936) has seen this structure too butdoes not describe it as an opening. The results of my feeding experiments(p. 292) are in favour of Brown's and Pitelka's conclusion, and the termcytostome will therefore be used to denote this opening. The cytostomehas a thickened anterior margin described by Brown (1930), Chadefaud(1938), and Hollande (1942) as a third, the falcate rod. As it is not connectedwith the two parallel rods and does act together with them during feeding,it is perhaps better regarded as a stiffened rim of the cytostome than as partof the rod-organ (Text-fig. 2, A, B).

Hall and Powell (1928) and Hall (1934) deny the existence of a second

Chen—Biology of Peranema trichophorum 283

opening, but some of their figures (1928, pi. I, 1, 2, 3, 4) indicate the presenceof two openings in the same individual. In their figures an opening at theanterior end of the body is connected by a canal to a second opening, labelled'cytostome1.1 also saw two openings but found that at the anterior end to leadinto the reservoir from which the flagella protrude, while the other (posteriorand ventral to the reservoir opening) is the true cytostome. In the figures ofLackey (1933) the cytostome is connected with the part of the reservoir canalin front of it, thus giving the appearance of a 'tuning-fork shaped cytostome'.

TEXT-FIC. 2. Peranema, Schaudinn/Heidenhain. A, viewed from ventral surface, B, twistedcell, viewed from the side.

Neither in living nor in stained specimens could a canal leading from thecytostome into the body be traced. It seems that there is not a permanentgullet, and that food enters through a temporary passage.

From the oblique position of the rod-organ it appears that bilateral sym-metry, so often given as a diagnostic character of the Peranemaceae, does not,in fact, exist in Peranema. Klebs (1883) seems to be the first who has usedthe term 'bilateral', but not in the correct sense: he meant 'dorsoventraP.Lemmermann (1913), Doflein (1916), and others followed him without beingaware of the inaccuracy. Dorsoventrality is very distinct in Peranema whichglides only on one surface, the ventral side of the body (cf. p. 301). Whenindividuals glide along the bottom of the container, they are observed throughthe dorsal surface with the posterior end of the rod-organ pointing towardsthe right as seen under a compound microscope (Text-fig. 9A, p. 299). Whenthey glide along the upper surface of the water, however, it is the ventral


surface of the body which is observed, and the posterior end of the rod-organpoints to the left (Text-figs, ic, 2A).

There are two flagella. The one directed forward is the one known aloneto most of the previous authors. It is wider than the second one and up totwice the length of the extended body. The second fiagellum is much narrowerand only two-thirds the length of the body. It turns back immediately afterprotruding from the reservoir opening and adheres to the ventral body surface,simulating a streak on the periplast (Korshikoff, 1923; Lackey, 1929, 1933;Hall, 1934). This second fiagellum is visible in living specimens under darkground illumination or under the phase-contrast microscope. By gentlepressure or by treatment with osmium tetroxide it can be detached. Skuja(1948) doubts the existence of the second fiagellum and believes that what isconsidered as a second fiagellum may only be a daughter fiagellum of adividing individual. It seems unlikely, however, that a newly grown fiagellumshould adhere to the periplast or that it should be narrower than the primaryfiagellum.

In stained specimens the two flagella are seen to originate independentlyfrom two basal granules which lie side by side in the wall of the reservoir,as in other Euglenineae. There is no bifurcation of flagellar roots. WhatDoflein (1916) and Brown (1930) describe as bifurcation seems to be a mis-interpretation due to failure to observe the second fiagellum. The structureof the flagellar apparatus of Peranema therefore accords with the views ofPringsheim (1948) regarding the flagellar apparatus of the Euglenineae.

Save for the difference in size, the anterior and posterior flagella have thesame structure. Each is differentiated into an axial core and an outer sheathwhich can be observed under the light microscope, particularly after treat-ment with Loeffler's stain, under dark ground illumination, or under theelectron microscope. The latter reveals still finer structures (Text-fig. 3A, a).Contrary to Lackey's statement, but in accordance with Hollande (1942), thesheath covers the whole length of the anterior flagellum except for the partwithin the reservoir (the root) which, when squeezed out by the contractionof the body during the preparation of specimens, is seen to be naked (Text-fig. 3B a). The sheath seems to consist of numerous fibrils radiating from theaxis, thus giving the whole flagellum the appearance of a test-tube brush.It is in this respect different from the flagellum of Euglena where the sheathconsists of longer fibrils wound spirally round the axis (Brown, 1945, and ownelectron-micrographs).

The axial core shows in gold-shadowed specimens longitudinal striation,indicating fibrillar differentiation. The number of fibrils, as revealed by theelectron-microscope, is considerably higher than the two of Brown (1945,1946) or the four of Dellinger (1909). The same has been found in severalother Euglenineae, including Euglena gracilis (Brown's material). My con-clusions are in agreement with Korshikoff's (1923) and those of Schmitt,Hall, and Jakus (1943) that the axis consists of a relatively large number ofthin fibrillae. The two thick fibrils, found by Brown in Euglena gracilis and

Chen—Biology of Peranema trichophorutn 285

'Astasia Klebsii' (in fact the colourless race of E. gracilis), may be bundles offibrillae situated in the edges of the ribbon-shaped flagella, as suggested bymy electron-micrographs.

By the application of protein-dispersing reagents an attempt was made torender the inner structure of the flagellum better recognizable. With previouslyfixed material no results were obtained, but the treatment of unfixed indivi-duals with chemicals, such as urea, known to disperse fibrillar proteinsstabilized by electro-valencies, constantly leads to the separation of theanterior flagellum into three fibrils, possibly as a result of the dissolution ofthe sheath. While in some of these reagents, e.g. urea (from saturated to3 M solution), NaOH (o-1-0-2 N), &c, the fibrils ultimately dissolve, inNaaSO3 (2-8 per cent.), dissolution is only partial even after 24 hours. InHC1 (0-2-5 N), on the other hand, the flagellum remains intact, even aftertreatment for 24 hours.

Like Deflandre (1934a, 1934*), who used nigrosin, and Hollande (1942),I could not find in Peranema the lash-like processes which, according to Vlk(1938), should be present on the surface of the flagella of all members of theEuglenineae. Neither Loeffler's mordant staining technique, nor electron-microphotographs showed any trace of such appendices, although bothmethods were successful in various species of Euglena, Trachelomonas, andPhacus (Pringsheim and Hovasse, 1950).

CULTIVATION AND GROWTH

Peranema was cultivated by Lackey (1927) in a wheat-water medium andby Hall and Powell (1927, 1928) and Mast and Hawk (1938) in a beef-suetinfusion, all with bacteria as food. Brown (1930) maintained that a culture ofPeranema will not live unless inoculated with some euglenoid flagellate, butShettles (1937) used a balanced salt solution with rice grains and Chilomonasas food.

1. Preliminary experimentsThe material with which I started had been grown for some time with

Saccharomyces exiguus as food suspended in half-saturated calcium sulphatesolution, a method which had previously been devised for Paramecium(Pringsheim, 1928). Cultures were fed daily during the first week, and lessfrequently later on. Flourishing cultures were obtained after about a month.Wheat and hay infusions and various bacteria from agar slopes did not supportsatisfactory growth.

With Saccharomyces exiguus as food the following solutions, apart fromcalcium sulphate, were tried: half-saturated aqueous calcium carbonate;diluted Benecke solution; one-tenth diluted natural and artificial sea-water;soil extract and glass-distilled water. Peranema multiplies in all these solutions:least well in distilled water, best in soil extract and almost as well in Beneckesolution, which both accelerate the rate of multiplication compared withcalcium sulphate solution, although they do not give a denser peak population.


TEXT-FIC. 3A. Electron-micrographs of the flagella of Peranema. a, curved anteriorflagellum; b, posterior flagellum; osmium tetroxide. x 12,000.

That Peranema can grow in distilled water does not imply that it needsno mineral salts, since yeast present in the culture supplies a certain amount.This is borne out by the facts that the health of the culture is poor in thebeginning but improves after repeated feeding and that without feedingPeranetna soon dies in distilled water but can live several days in solutionswith calcium (cf. Pringsheim, 1928, for Parameciutn). Accordingly additionof very low concentrations of calcium salts greatly improves the cultures,

TEXT-FIG. 31J. (a) root of anterior flagellum squeezed out of reservoir; (b) end of anteriorflagellum showing core and sheath; (c) end of posterior flaRellum; sheath much less dense.

X 16.000.

288 Chen—Biology of Peranetna trichophorum

one-sixteenth to one-sixty-fourth being better than more concentratedsolutions.

Apart from Saccharomyces exiguus the following organisms from purecultures were tested as food: Polytoma uvella Ehrenb., ChlorellapyrenoidosaChick, Prototheca Zopfii Kriiger, Stichococcus bacillaris Naeg., ChiUmonasparamecium Ehrenb., Euglena gracilis Klebs, Astasia longa Pringsheim,Saccharomyces cerevisiae Hansen, and Rhodotorula glutinis Fresenius. OnlyS. cerevisiae is as good as S. exiguus in maintaining Peranema. Rhodotorula,taxonomically near to it, may be inferior because it cannot be suspended inwater. Chlorella in the light grows so fast that Peranema is eventually sup-pressed. The two species of Euglenineae as well as Stichococcus only supportmoderate growth. Between a green Euglena and Peranema there is a com-plicated balance in multiplication, the former first growing quicker, thenPeranema catching up, until eventually, when the food organism declines,the latter also gradually decreases until after about a year only a few individualsremain.

The influence of the pH has been investigated by using a series of Clark'sbuffer solutions ranging from pH 4 to 9. With S. exiguus as food Peranemawas found to multiply between 5-2 and 8-4, with an optimum at neutrality.In order to see whether Peranema is capable of becoming adapted to higherconcentrations of mineral salts, dilutions of artificial sea-water ranging from1/20 to 1/4 were inoculated with Peranema. The growth was normal in thelowest concentration, and there was none in 1/5 and 1/4. If inoculated from1/20, growth occurred up to 1/5. Even if the increase of concentration waseffected by gradual evaporation of a culture in 1/20 sea-water, Peranemacould tolerate only a concentration of 1/4-2 (24 per cent, sea-water). Com-pared with other flagellates (Finlay, 1930; Hardin, 1942) Peranema has onlya very restricted capacity of adapting itself to salt-water.

2. Bacteria-free cultures

Peranema purified by washing (Pringsheim, 1946) could be grown withSaccharomyces as food in two-member pure cultures. Healthy cultures were,however, only obtained with soil extract as the medium. No growth occurredin dilute sea-water or calcium sulphate solutions, even after repeated inocula-tion. As both these media support growth when bacteria are present, bacteriaevidently play some part in the nutrition of Peranema, although they alonedo not suffice to maintain the cultures. Butterfield (1929), Lilly (1942), andHardin (1944) had similar results with various other protozoa. Since soil-extract works well, whether bacteria are present or not, it seems that somesubstances produced by bacteria and beneficial to the growth of Peranemacan adequately be supplied by soil extract. Attempts to cukure Peranema onyeast killed by heat, even as low as 8o°, failed.

Really pure and thriving cultures were obtained with cow's milk. One dropof milk was added to 8 c.c. of either distilled water, calcium sulphate solution,or 1/10 sea-water. The resulting mixtures were heated in a steam-chamber


for an hour on three consecutive days, and inoculated with single, washedindividuals. Multiplication in all three media was so intense that after 3 weeksthe inner surface was densly covered with Peranema—large, healthy indivi-duals containing numerous food vacuoles. Tests proved the sterility of all thecultures. No difference in the rate of multiplication in the three media hasbeen observed. For maintaining the cultures, however, that with calciumsulphate is preferable to the others because during sterilization the milk iscoagulated in this medium, and the fluid is then clear, permitting betterobservation of the organisms. Otherwise the milk emulsion remains turbiduntil the suspended milk droplets have been ingested by the Peranema.

Milk cultures decline earlier than those with yeast as food, ceasing toflourish after 3 months owing mainly to the accumulation of waste products.The decline could not be prevented by adding more milk. Autoclaved milkgave good results initially but not in successive subcultures. Somethingessential for Peranema seems to be destroyed by excessive heating. Theoptimum concentration of milk is 0-5-1 per cent. Concentrations higher orlower than these do not support appreciable growth. Bacto peptone, Bactotryptone, yeast extract, glucose, starch, and almond-oil have no effect whenadded to milk cultures, whereas 0-1-0-2 per cent, beef-extract acceleratesgrowth, although it does not increase the final density of the population.

With milk as food Peranema is not only bigger than with yeast (cf. p. 282)but also contains more and larger paramylon grains and oil-droplets, and itmultiplies more quickly. In milk cultures the population reaches its maximumof about 17,000 individuals in 1 c.c. after about 1 month. This density ismaintained for about 10 days and then falls steadily to a level of about12,000 individuals which lasts about 50 days; subsequently the populationdeclines rapidly. Yeast cultures reach only a maximum of about 7,000-8,000in 1 c.c'. but last longer, and extinction does not occur until after 10 monthsor more, provided food is added repeatedly.

Fairly good results were also obtained with egg-yolk as food, although thedensity of the cultures was never as high as in milk cultures. Mixtures ofcasein and butter have been tested in an attempt to analyse the effect of theingredients of milk. Fat-free casein was kindly supplied by Dr. E. Kodicek(Dunn Nutritional Laboratory, Cambridge). To 10 c.c. of distilled water,calcium sulphate solution, soil-extract or o-i per cent, beef-extract was addedo-i per cent, butter plus 0-02 gm. casein. Culturing and subculturing waspossible, particularly in the last two media, but none of the cultures wasnearly as good as those with milk. Addition of whey, prepared by clottingfresh milk with commercial rennet, gave but little improvement. Butter oran ether extract of milk, both in o-i per cent, beef-extract or yeast-extract,as well as casein in soil-extract, beef-extract, or yeast-extract all supportmoderate growth. Addition of glucose does not improve the results. Theoptimum concentrations of butter and casein are 0-36 and 0-2 per cent,respectively. On the whole none of these media is satisfactory. Since butteralone does not support growth it seems that Peranema can multiply well only


when some proteinic substance is present which is digested inside the body.No evidence of proteolytic enzymes being excreted could be found by placingdrops of a dense Peranema culture on gelatine slopes under aseptic condi-tions.

Many attempts to cultivate Peranema in monophasic liquid media have beenmade. Of the 45 mixtures tested—various combinations of beef-extract,yeast-extract, liver-extract, tryptone and a number of other peptones invarious degrees of hydrolysis, glucose, alkali caseinate, and sterile filtrates ofcasein solutions, egg-albumin and blood-serum—none supported growth. Sofar as these results go, Peranema seems to be strictly zootrophic, as it requiresparticulate protein food.

FEEDING HABITS

While the feeding habits of ciliates and rhizopods have been extensivelystudied, little is known about those of zootrophic flagellates. Peranema isrelatively large in size and slow in motion, and ingests readily a great varietyof food particles. It seemed therefore suitable for observations on behaviourtowards food substances.

In the first place an answer was sought to the question whether there is anyfood selection, and experiments were begun in which they were fed: (1) withdifferent kinds of substances separately, to see if there is variation in thefrequency of ingestion; (2) with a mixed diet, to see if Peranema has theability to select one and reject the other component; and (3) with non-nutritive coloured substances, continuously for a longer period, to see ifPeranema can reject these.

A few drops of a Peranema culture, containing about 4,000 individuals,were transferred to watch-glasses with a suspension of food-particles or oforganisms. At intervals samples were counted in order to determine the ratioof the number of individuals with, to that of individuals without, the food inquestion. The result obtained was that, among the substances used, caseinwas ingested most frequently, while sand-grains and calcium carbonatepowder were not taken at all. Between these two extremes, arranged in orderfrom higher to lower frequency of ingestion, were: carmine, Chlorella pyre-noidosa, Stichococcus bacillaris, Saccharomyces exiguus, Chinese ink, Euglenagracilis, E. viridis, and starch-grains. Thus carmine and Chinese ink, both ofno food value, are more frequently ingested than both species of Euglena,on which Peranema can live.

Carmine was ingested also in the presence of a food substance, for instancecasein, Stichococcus, or Saccharomyces. Like carmine, casein was ingestedmore readily than any of the organisms tested in mixture with it. Carmineparticles are ingested even when the individuals have already taken a con-siderable amount of real food, e.g. Euglena or casein. Satiety does not causePeranema to reject carmine, unlike Stentor (according to Schaeffer, 1918).Even by prolonging the supply of carmine, Peranema cannot be 'trained' toreject it, as Metalnikow believed to have proved in Paramecium.

Chen—Biology of Peranema trkhophorum 291

What there remains of food-selection in Peranema has nothing to do withthe food-value of the substances, but is determined by physical and chemicalproperties and, in the case of living organisms, by the motility of the prey.

The controversy regarding the mechanism and the place of ingestion hasalready been mentioned (p. 280). Previous observers had mostly drawn theirconclusions from morphological observations (Hall and Powell, 1928; Brown,1930; Lackey, 1933). It was hoped that feeding experiments would help toclear up both these points. In observing the process of ingestion, hanging-drop preparations were used containing individuals which had been without

TEXT-FIG. 4. Successive stages (A-D) of Peranema swallowing a Euglena viridis. The wideextension of the anterior end is seen, and also the independence of the food entrance from

the reservoir.

food for a day. Of the thirteen species used as food Euglenineae of larger sizegave the most valuable results. In no case were actively moving formsingested.

When 10-20 individuals of Euglena viridis were added to a hanging drop,some soon became motionless and were then attacked by Peranema. Themoment the tip of the undulating anterior flagellum of a Peranema touched aEuglena, the whole flagellum began to beat violently; whereupon the cell-bodycontracted and the flagellum drew back. Soon the cell relaxed and the flagellumresumed normal movement. The process of contraction and recovery wasrepeated several times until the body came into contact with the Euglena,when ingestion began. It always started at the anterior end of the prey (Text-fig. 4). When a Peranema touches the front end of a Euglena, the rod-organtogether with the adjacent region of the ventral body-surface moves forwardfrom its original position, becoming level with the anterior end, until ittouches the Euglena. The rod-organ then protrudes a little and becomesattached to the surface of the Euglena. The body of the Peranema starts tomove forward around the prey, while the rod-organ remains attached to theperiplast and begins to push the Euglena through the widely expanded anteriorend of the Peranema into the interior. The rod-organ is then detached from the

292 Chen—Biology of Peranema trickophorum

periplast, moves over the surface of the Euglena and is re-attached on anotherpart of the periplast, while the body of the Peranema follows. By repeatedlyattaching, pushing, detaching and re-attaching the rod-organ, with forwardmovement of the body, the Euglena is swallowed. The time required for thewhole ingestion varies from 2 to 15 minutes, with an average of 8 minutes.During ingestion the two rods always move together as if connected.

It is not uncommon for a Peranema to ingest two Euglenae at a time,although its size is scarcely larger than that of the prey. A Peranema was seen,

TEXT-FIG. 5. Peranema cutting open the periplast of Euglena viridis and ingesting its proto-plasm, A, Peranema protrudes into the cell of Eugletia after perforating its periplast with itsrod-organ, B, TWO individuals attack the same Euglena. The protoplasm of the Euglena issucked in by one of the Peranemae through a way separate from the reservoir, which remains

clear, c, Euglena, after being attacked, shows three splits on the body-surface.

2 minutes after taking in a Euglena, to meet another which was then attackedand swallowed immediately. Both of the two ingested Euglenae were soondigested. In some instances the second Euglena was only partly swallowedand, perhaps because there was no room to spare for it, eventually egested.

Sometimes a Peranema, instead of ingesting a Euglena as a whole, cuts openthe periplast and sucks out the contents (Text-fig. 5). When the cytostomeis applied to the attacked Euglena, the rod-organ is protruded and rasps upand down over the periplast of the prey. After 6-10 minutes the protoplasmof the Euglena with the green chromatophores begins to flow into the bodyof the Peranema, not through the reservoir but through a passage lateral andnearly parallel to it (Text-fig. 5B). This passage can only be seen when foodis passing and may, therefore, be only a temporary, rather than a permanent,structure. The reservoir and its canal, during this process of ingestion, remainclear and free from food particles. Ingested food is accumulated at the posterior

end of the passage to form digestive vacuoles which are detached appa ltlyby contractions of the body. The shape of these vacuoles is usually that ofthe food enclosed, i.e. the food appears to be in direct contact with thesurrounding cytoplasm.

Chen—Biology of Peranema trichophorutn 293

Sometimes Peranema sucks in only part of the contents of a Euglena; inother cases it consumes the whole, leaving only the periplast. As the contentsof the prey decrease during the sucking, the anterior end of the Peranemamay penetrate through a hole in the periplast into the interior and continueto suck there (Text-fig. 5A). The rod-organ, protruding slightly into thecytoplasm of the Euglena, seems to help in pushing the food into the body.Sometimes the rod-organ moves slowly along the boundary between thecytoplasm and the periplast, separating the one from the other. The remainingempty periplast shows regularly one or more splits where the rod-organ hasbeen applied (Text-fig. 5c).

The frequencies of the two types of ingestion, engulfing and sucking, varywith the circumstances. When a Euglena viridis is attacked by a singlePeranema, the whole cell will be swallowed. When it is attacked by two orthree, it will be either shared by the predators, each sucking a portion of it,or engulfed by one of them. When it is attacked by many Peranemae, it isalways pierced and sucked by a number of individuals; swallowing by a singleindividual has not been observed under such conditions. Towards Euglenagradlis and its apochlorotic race ('Astasia longa'), Peranema behaves in thesame way as towards E. viridis.

Euglena spirogyra, although much larger than Peranema itself, is alsoattacked, but only when it has been killed, e.g. by pressure or by heat. It iseasily killed by pressing the coverslip, and cytoplasm flows out through lesionsin the periplast. If then Peranema individuals are introduced, they soon attackthe wounded Euglena. Those which arrive first approach the wounds whencecytoplasm is flowing. Only when such sites are occupied do they begin to cutnew openings with the rod-organ. Heat-killed E. spirogyra is preferentiallyattacked at the anterior end of the cell. The front part of Peranema, truncatedand widely expanded, is applied firmly to the surface of the prey, while therod-organ moves to cut the periplast. It was observed that, while the cytostomeand its attached rod-organ were working in contact with the periplast of theEuglena, the anterior end of the Peranema bearing the flagellum and thereservoir opening was shifted to one side, i.e. out of contact with the Euglena(Text-fig. 6A). The cytoplasm of the prey could be seen flowing through thecytostome into the body while the reservoir remained empty.

When a sucking Peranema becomes full of food vacuoles it detaches itselffrom the Euglena and its place is taken by another individual. As the cytoplasmof the prey gradually decreases in volume by continuous sucking, the Peranemapenetrates more and more deeply into the interior and may come to lie com-pletely within the almost empty body of the prey. In one instance threeindividuals were seen within the periplast of a single E. spirogyra.

If not more than four individuals attack a E. spirogyra the one nearest thefront end usually tries to engulf it, although this can never be successfulowing to the smaller size of Peranema. The attack proceeds in the same manneras that on E. viridis and, after a few minutes, the anterior end of the E. spiro-gyra sinks into the body of the Peranema through its tremendously expanded


anterior end (Text-fig. 6B). When the Peranema has eaten to capacity, onlyabout one-fifth of the prey is swallowed. As the Peranema attempts to engulfmore, the rod-organ moves here and there attempting to anchor itself to theperiplast, and the body rotates about the axis of the Euglena.

It is at this stage that the ingested part of the Euglena, sunk deeply into thebody of the Peranema, is clearly seen to be outside the reservoir, whichremains empty and transparent and is pressed to one side of the body (Text-fig. 6B). Eventually the Euglena is egested through the cytostome. Immediately

TEXT-FIG. 6. Peranema feeding on heat-killed Euglena spirogyra. A, during the attack;reservoir and flagellum shifted away from the prey; rod-organ seen to be independent of thereservoir. B, Three individuals attacking the same Euglena; the one in front is trying to engulfthe whole prey, the reservoir remains empty; the other two begin to apply their rod-organs,c, The same individual (i in B), immediately after egesting the partly swallowed Euglena;note the independence of the rod-organ and the cytostome in front of it, from the reservoir;

the space, which was previously occupied by the prey, is separated from the reservoir.

after this, while the anterior end of the Peranema remains for a short timewidely expanded, the independence of the feeding apparatus from thereservoir and its canal is especially clear (Text-fig. 6c). The cytostome, sinceit has not yet contracted to its normal size, can be observed near the distalend of the rod-organ as a crescent-shaped opening lateral to the reservoir.The space in the body of the Peranema previously occupied by the ingestedpart of the Euglena remains as an empty vacuole not connected with thereservoir system. Soon the anterior end is restored to its normal taperingshape, and the cytostome with its attached rods returns to its previousposition, ventral and posterior to the reservoir opening, and regains its formerappearance as a transparent region near the end of the rod-organ.

Cyclidiopsis acus Korschikoff (—Astasia Hnealis Pringsheim) is much lessattractive to Peranema than Euglena spirogyra, but otherwise affects it in asimilar way.

Cannibalism occurs in Peranema. When transferred from a culture to a


hanging drop, a certain number of individuals are usually killed or injuredand shed their flagella. They are soon eaten by the active individuals, theirperiplasts being perforated and the contents sucked out. No swallowing ofthe whole organism has been observed.

Chilomonas paramecium is only ingested when motionless, and is theningested as a whole, without its surface being perforated. Contrary to thestatement of Hall (1933) Chilomonas was never found to pass through thereservoir system. Hall relied on the observation that in his stained preparationsthe food vacuole containing the ingested Chilomonas appeared to be connectedwith the reservoir. This, however, does not necessarily mean that the preyis ingested through the reservoir. A food-vacuole with Chilomonas occupiesa relatively large space, and when the cytoplasm contracts during fixation,the vacuole may seem to be connected with the reservoir.

Cells of Saccharomyces exiguus and S. cerevisiae are always swallowed whole.For the purpose of observation yeast cells were made more visible by stainingwith 0-2 per cent. Congo-red solution. When a gliding Peranema touches ayeast cell with its flagellum it either contracts and moves away or continuesgliding, so that the yeast cell 'flows' along the ilagellum to the anterior end ofthe body. When it arrives there Peranema reacts in various ways. It eitheringests it or keeps gliding normally, leaving it behind. When ingestionoccurs, Peranema usually contracts considerably so that, by the time it recoversto its normal shape, the yeast cell is already inside the body. Occasionally,however, the yeast cell is ingested while the Peranema maintains normalgliding, without contortion of the body. In the latter case, the passage of theyeast cell into the body can be observed. The observation confirms that itpasses from the cytostome into the cytoplasm not through the reservoirsystem, but through a way near to but not connected with this. Sometimes,however, when the ingested cell lies ventral to the reservoir it seems to bewithin it, and when it passes posteriorly, it may give the impression that itcomes out from the end of the reservoir. A side view will reveal the trueposition of the yeast cell, ventral to and outside the reservoir. The rod-organappears to be of little importance in the ingestion of such small organisms.From one to six yeast cells have been observed in a single food-vacuole.

These observations confirm the conclusions drawn from morphologicalstudies described above and the statements by Brown (1930) and Pitelka(1945). They are also in agreement with what is known in other flagellates.In no case have particles been seen to enter the reservoir of any of the Eugleni-neae, nor indeed of any of the other flagellates. Kent's (1880-2) and Tann-reuther's (1923) claims that Euglena ingests particles through its reservoirhave been proved to be wrong (Mainx, 1928; Hall, 1933ft). Apparently thereservoir system has some other function than feeding (e.g. respiration ordrainage) and when, in the evolution of the Peranemaceae, the mode ofnutrition changed to zootrophy, a new opening was developed for the purposeof ingestion. If that be the case, the terms 'cytostome', 'gullet', and 'pharynx'should not be used for the parts of the reservoir system.


DIGESTION

Almost nothing is known of the digestive powers of non-parasitic flagellates,although proteolytic enzymes have been found in cultures of certain speciesof Euglena (Mainx, 1928; Jahn, 1931; Hall, 1937).

Which of the substances present in the food eaten by Peranema are indeeddigested can only be ascertained by feeding experiments with different kindsof food. For this purpose Peranema from cultures with yeast as food has beenused. Since such individuals contain only a moderate amount of reservesubstances, observation is relatively easy. The food substances used were:lipoid-free casein, egg-albumin, almond-oil, and rice-starch. To one of twowatch-glasses with 2 c.c. of the culture-fluid food was added, while the otherone was used as control. At intervals samples were taken for microscopicexamination. Hanging drops containing single individuals, which were isolatedafter having ingested a number of food-particles, were also used. These gavea clearer picture of the changes in the ingested food, because the uptake ofnew food-particles was prevented. Simple histochemical tests (such as iodinefor starch; Sudan III, sudan black, and chlorophyll-extract for fats; potassiumhydroxide for paramylon) were made.

The food-vacuoles of Peranema assume the shape of the ingested food anddo not undergo translocation along a constant path within the cytoplasm.They either remain at the same place or are moved at random by body-movements. They are largest when newly formed and decrease in sizegradually until they are entirely absorbed; or, more usually, the remains,much reduced in bulk, are discharged through the area of defaecation.Although the digestible parts of the food must have been liquefied in thecourse of digestion, an enlargement of the food-vacuoles, such as commonlyoccurs in ciliates and rhizopods, has never been seen.

The digestion of Euglena viridis by Peranema may be taken as an example(Text-fig. 7). Newly ingested cells preserve their colour for about half anhour and then fade to yellow; no change of size is as yet observable. At thesame time a very thin layer of the cytoplasm of the Peranema around theEuglena becomes transparent. After 1 hour the size of the Euglena hasdecreased to about two-thirds. The transparent layer is still visible. By thenext day the Euglena has been reduced to about one-fifth its original size.Its shape has become ovoid, its colour brown, and it is no longer recognizableas a Euglena. The transparent layer has disappeared, and many paramylongrains are seen in the cytoplasm of the Peranema. The remains of the Euglenaare usually discharged, but sometimes they may remain in the body foranother day or two, although no further decrease in size can be detectedduring this time. The discharged remains seem to consist mainly of theperiplast. Striations are still visible, as well as splits, apparently those madeby the rod-organ.

By feeding Peranema with yeast cells stained with indicators (Shapiro,1927, &c.; Pringsheim 1928), the pH of the food-vacuoles was found to lie


between 5-8 and 6*8. This is very different from those in ciliates and rhizopods,where the pH can be as low as 3 (Shipley and de Garis, 1925; Shapiro,1927; Pringsheim, 1928; Mast, 1942; Mast and Bowen, 1944).

While rhizopods and ciliates are known to digest fat (Lund, 1914; Daw-son and Belkin, 1928, 1929; Mast, 1938; Wilber, 1942) nothing so far is

TEXT-FIG. 7. Digestion of Euglena viridis by Peranema. A, Peranema engulfing a Euglena.B, The Euglena is ingested; note the two splits on the surface of the Euglena made by the rod-organ, c, After an hour, the size of the Euglena has diminished and a thin clear layer appearsaround it. D, After 2 hours of ingestion. E, The next day, the size of the prey is much reduced,and the clear layer has disappeared; paramylon-granules appear in the cytoplasm of thePeranema. F-H, Successive stages on the third day. H, The remains of the Euglena aredischarged. I, Enlarged view of the discharged Euglena with periplast-striation easily visible.

known in this respect about flagellates. For the relevant experiments almond-oil, stained dark blue with sudan black, was used as food, in order to distin-guish the ingested fat from that formed by resynthesis. Peranema ingestedthese stained oil-droplets without visible ill effects (Text-fig. 8). After 2 hoursof feeding almost every individual contained a number of blue food-vacuoles.The sudan III test did not reveal any increase in the number of oil-dropletsin the cytoplasm. After 5 hours, however, in addition to the large, spherical,blue oil-drops, many droplets, staining red with sudan III, were present inthe cytoplasm of most individuals. Five hours later the size and number ofcolourless droplets stainable with sudan III had markedly increased. Inindividuals isolated in hanging drops it was found that, while the secondary


droplets increased gradually, the ingested oil-drops decreased in size.Evidently these colourless droplets are fat-substances resynthesized fromproducts of digestion, rather than formed by splitting up the ingested oilinto smaller droplets.

TEXT-FIG. 8. Digestion of almond-oil, A, Control individual not fed with almond-oil, B-D, Indi-viduals which have taken up almond-oil stained blue with sudan black supplied in a watch-glass. B, After 2 hours of feeding, c, After 5 hours. D, After 24 hours of feeding. E-c, Successivestages of individual isolated in hanging drop and digesting ingested oil. E, TWO drops ofalmond-oil have just been ingested. F, After 3 hours; the drops have already decreased in size.G, After 5 hours of ingestion; many oil-droplets have appeared in the cytoplasm. H. Individualafter 24 hours of digestion, with a further increase in the number of oil-droplets in the cyto-plasm ; the remains of one of the ingested oil-drops (d) have been discharged into the hangingdrop./ = food-vacuoles, containing dark-blue oil-drops; o = colourless oil-droplets stainable

with Sudan III; p — paramylon granules.

Starch is also digested by Peranema, though but slowly (Text-fig. 9). After1 day's feeding with rice-starch many ovoid paramylon granules appear inindividuals which before contained only a few small granules. The newlyformed granules are of the same size and shape as those in individuals frommilk cultures. After some time, only the centre of the ingested starch-grainsstains blue with iodine, while the outer layers are faintly brown. This indicatesthat the starch is chemically attacked. The decrease in size of the starch grainsis clearly observed in hanging-drop preparations. The correlation betweenincrease in number and size of paramylon granules and the decrease in size


of the ingested starch shows that the former result from digestion of thelatter.

For observations on the digestion of protein, casein was used, mainlybecause it is easy to handle and has proved a suitable food (Text-fig. 10).It was found that after 24 hours, when the ingested casein particles have

zJTEXT-FIG. 9. Digestion of rice-starch, A, Control individual without starch. B and c, Indi-viduals which have taken up rice-starch in a watch-glass. B, After i hour of feeding; starch-grains stain dark blue with iodine, c, After 34 hours of feeding, showing the appearance of

vi t* oiai.ivii-gi.aii*. a—\Jt L>UIA>\.NIVL aLa^i.3 ui aii uiuiviuuai touiaicu 111 a naH£lH£ QTOp SltCf lfl^CSt-

ing four starch-grains. E, Immediately after feeding. F, After 2 hours of digestion, showing clearlayers around two food-vacuoles. G, After 8 hours: paramylon-grains have augmented; onestarch-grain stained only faintly in its peripheral layer while the centre was dark blue.H, After 24 hours: many paramylon-grains, starch gradually disappearing, p = paramylon-

grains ; j = ingested starch; s' = starch blue by iodine; s" = faintly stained outer layer.

decreased to about one-half or one-third of their original size, many oil-droplets of various size appeared; the quantity of paramylon only increasedsubsequently. In order to decide whether the oil is derived from fatty impuritiesin the casein, specially re-purified casein was used in repeating the experiment.The result was almost the same. It is concluded, therefore, that Peranema iscapable of digesting protein and of converting it to oil and paramylon.

Both oil and paramylon are to be considered as reserve food, because inflourishing cultures, individuals contain more of them, and when the culturesare declining, both decrease in quantity until very little is left, while the size


of the cells decreases also. The same changes were observed in indivispecially starved. During one day of starvation the number of oil-drcincreases considerably, but after that both oil-droplets and paramgranules decrease steadily in number and size until the individuals die.

TEXT-FIG, IO. Digestion of casein. A, Control individual not provided with case-i. „,Individual fed with heat-denatured but otherwise untreated casein for 24 hours in a watch-glass, showing the appearance of paramylon-grains and many oil-droplets, c, Individualsupplied with alcohol-ether extracted casein, after 24 hours, showing a lesser increase inoil-droplets, D-F, Successive stages of an individual digesting extracted casein after isolationin a hanging drop. D, Five food-vacuoles with casein, E, After 2 hours of digestion, showinga thin, clear layer around some of the food-vacuoles. F, After 24 hours, showing decrease insize of casein-particles and increase of paramylon-granules; scattered between them are oil-droplets staining with sudan III. c = ingested casein; / = oil-droplets; p = paramylon-

granules.

diminution of the Peranema cells is considerable, from the average size ofindividuals in yeast cultures, 50-55 X 16-18/*, at the beginning, to 42-46 X9-10/u. on the seventh day of starvation. Shortly after that they all perish.

MovementMOVEMENT AND CHEMOTAXIS

It has long been known that Peranema is capable of performing two kindsof movement, gliding along the substratum or the water surface, and swimmingfreely in the water (Kent, 1880-2; Mast, 1912; Massart, 1920). AlthoughMast has observed both gliding ('crawling') and swimming movement, he


believes that the latter occurs only exceptionally. I have regularly observedfree-swimming individuals in undisturbed cultures, however, and can testifythat swimming occurs under natural conditions although not as frequentlyas gliding.

The mechanism of the movement of the Peranema flagellum has often beenstudied (Biitschli, 1883-7; Gray, 1922, 1931; Krijgsman. 1925; Lowndes,1936, 1941, 1944a, b; Brown, 1945). But the nature of the gliding movementhas not attracted much attention. In the following the term 'flagellum' isused for the long, free Hagellum. The function, if any, of the posteriorflagellum, attached to the ventral body surface, is unknown. As stated byMassart (1920), Peranema always glides on the same (ventral) surface, whetherover solid bodies or the water-air interface. During gliding the Hagellumstretches out in a straight line and appears rigid and motionless save at thedistal end, which beats incessantly to the right as seen under the microscope,its tip describing roughly an ellipse. The change of direction of movementseems not to be determined by the action of the flagellum. In most of myobservations the body turned first to the new direction while tbe flagellumremained behind; only in a few cases did the flagellum bend first. Th« sameconclusion has been reached by Mast (19x2).

Mast holds that Peranema glides more quickly when the undulation ofthe flagellum extends over a greater length as a result of irritation. This wasnot confirmed. On the contrary, when the individuals are stimulated, asVerworn and Lowndes (19446) have already pointed out, though the flagellumbeats then throughout its entire length, the body contracts and no longermoves forward. Maximum speed of gliding can only be observed when boththe flagellum and the body are in a state of full extension. Measurements atroom temperature have given an average speed of i5*6/u./sec. when indivi-duals are moving along the bottom of the container, and 22-9 ̂ /sec- whenthey are gliding over the water-air interface. Mast (1912) recorded a speedof 2i>7-43'4/*/sec, Shortess (1942) 26-17/x/sec, and Lowndes (1944^)20/i/sec, all without mentioning the substratum over which the individualsmoved. No correlation has been found between the size and speed of theindividuals.

Gray (1931) and Lowndes (1936, 1941, 1944a, b) believe that the flagellumis an active unit capable of generating its mechanical energy. The latter authorstated that the undulation wave starts from the base and is transmitted to thetip. This was confirmed by studying the movement in a soft 0-25 per cent,agar jelly. The fact that only the tip of the flagellum beats strongly suggestsalso that the flagellum generates at least part of its energy. However that maybe, the role of the flagellum in gliding is by no means established. Duringgliding Peranema is attached to the substratum so firmly that it is not readilydetached by the sucking force of a capillary tube. Furthermore, whenPeranema is transferred to a new medium, it is usual for a number of indivi-duals to lose part of their flagellum. Normal gliding was observed in individualswith only two-thirds of the flagellum left, so that the active tip was no longer


present. In individuals with still shorter flagella, about one-third of the body-length, normal gliding movement no longer occurs, although they can proceedslowly by wave-like contractions passing from the anterior to the posteriorend of the body. Such wave-like contractions can occasionally be observedin normal individuals also. Evidently the cell-body is capable of movingby itself.

The results were confirmed by observations on individuals with theflagellum removed with the aid of a pair of glass needles under a dissectingmicroscope. When the whole flagellum was removed the individual imme-diately rounded up and remained motionless, as if dead. After not more than2 minutes it recovered and, while the body remained in the same place, theanterior end was extended from the mass and then withdrawn by contraction.This was repeated again and again. After 17 minutes a very short flagellum,not longer than one-fifteenth the body-length, had appeared; it could be onlyseen under high magnification. This short flagellum was for the most partinactive, but occasionally gave a few feeble beats. The individual was stillunable to glide. After about half an hour the flagellum had grown longer, toabout one-fifth of the body-length. By this time the body was capable of beingextended to its full length but never kept this shape for longer than a minute.After 50 minutes the flagellum had grown to one-third of the body-lengthand was beating forcibly, but stiffly, like a brandished stick rather than likea whip; the body now progressed apparently by peristalsis, waves of con-traction being seen to pass from the anterior to the posterior end of the body.

Only after 2 hours, when the flagellum had attained a length equal to thatof the body, its end began to undulate in the normal way, and gliding move-ment was resumed. The results obtained from individuals with only part ofthe flagellum removed were practically the same; i.e., the body is capable ofadvancing by peristaltic contractions while the flagellum is still short andseemingly functionless. Gliding, however, only begins when the flagellumhas reached almost its normal length, earlier after shedding the flagellumthan after its removal by an operation.

All these observations, especially that with half the flagellum shed, supportthe suggestion that the body is moving independently of the flagellum, whichis not essential for gliding. This is comparable to the behaviour of otherEuglenineae, e.g. Euglena mutabilis Schmitz, which are capable of glidingalthough devoid of actively functional flagella, only short stumps, not stickingout of the reservoir opening, being present. It is possible that there is inPeranema a correlation between the activity of the flagellum and the main-tenance of the normal shape of the body. Like other gliding organismsPeranema discharges mucilaginous matter which tends to cover the surfaceof the container of old cultures, even if free from other organisms. Whatconnexion this mucilage may have with the gliding movement is not knownin Peranema any more than in other instances.

Lowndes's assertion (19446) that the flagellum may have a sensory functionis in agreement with my observations. When a gliding Peranema hits an


obstacle with the tip of its flagellum the latter undulates strongly, and thebody contracts spasmodically before it has itself touched the obstacle. Whetherthis is a grain of sand or suitable prey does not at first make any difference.We conclude that the tip of the flagellum is mechanically irritable, and thatthe difference between inert particles and food only affects behaviour if thelatter produce chemotactically active substances which prevent the glidingindividual from turning away. When beginning to glide again, an individualthat has hit an indifferent object moves in any direction, without preference;one that has come near to a nutritive object, however, glides nearer andnearer to it. The flagellum is therefore sensitive- to touch, while chemicalsense may be located in the cell-body.

Whereas the role of the flagellum in the gliding movement is doubtful,swimming is definitely performed by its action. By using a compound micro-scope with the tube horizontal and fastening a test-tube culture" to the verticalstage, the swimming movement can be watched without agitating the culture.As the individuals proceed in all directions at random, the translocation iscertainly not due to streaming of the medium. During swimming the bodyrotates continually, usually counter-clockwise but sometimes also clockwise.Generally the posterior end moves almost steadily in a straight line while theanterior end describes a spiral, the body pivoting about the posterior end,but sometimes the body describes a double cone. The greater, basal part ofthe flagellum rotates, describing a funnel, also counter-clockwise or clockwise,while its tip is bent sideways and undulates as in gliding. Peranema does notswim steadily over a long, distance. At short intervals of time the body con-tracts without apparent cause and, after re-extending, the individual startsswimming again, either in the previous or more frequently in anotherdirection. The speed of swimming of different individuals varies between20-3 and 42/t/sec. with an average of 31/i/sec. (faster than when gliding).

Chemotaxis

The existence of chemotactic irritability in Peranema is revealed by thetendency to assemble around sources of food. When for instance a deadEuglena spirogyra is introduced into a drop of a Peranema culture, manyindividuals soon assemble around it, and when a piece of casein is hung ina test-tube culture of Peranema, it is densely covered with individuals aftera few hours. Chemotaxis is, however, not restricted to substances suitableas food. If coarse carmine lumps of the size of about half a wheat grain areadded to some drops of Peranema culture in a watch-glass, individuals soonassemble around them and proceed to ingest them, piece by piece. It isevident that carmine is not ingested by chance.

For testing chemotaxis the capillary method of Pfeffer (1888) was adaptedto the special needs of this organism. Pringsheim and Mainx (1926) in theirwork on Polytoma filled capillaries, left open at both ends, by their ownsuction force, instead of under the air-pump; but for the present experimentsit was necessary to seal one end after filling, by quickly dipping into melted


paraffin, because during the relatively slow movement of the organisms thefluid column in the capillary began to move. Another innovation connectedwith the larger size of the organisms was the use of watch-glasses instead ofdrops between slide and slip. About 0-2 c.c. of the stock-culture was pouredinto the watch-glass and left on the stage of the microscope for at least anhour before the open end of the capillary was dipped into its centre.

Very distinct reactions are exhibited towards acetic, nitric, hydrochloric,and sulphuric acids. At certain concentrations, for instance 1/100 n HC1,individuals assemble around the opening of the capillary, forming a ring witha clear space within, owing to equilibrium in this zone between attractive andrepulsive action. The higher the concentration, the larger the diameter of theempty space. When the concentration of the acid is decreased, the emptyspace becomes smaller and eventually, at proper concentrations, the indivi-duals react by directly coming to the opening of the capillary, forming acluster instead of a ring. Such concentrations are considered to be those towhich Peranema reacts positively. They have been found to be:

Acetic acid . . . . 1/200-1/500 NNitr ic acid . . . . 1/750-1/1000 NHydrochloric acid . . . 1/650-1/800 NSulphuric acid . . . 1/550-1/800 N

The fact that the thresholds of the three strong acids are of the same orderand are more dilute than that of the weak acid, and that corresponding saltsof these acids do not provoke any reaction of Peranema, indicates that thechemotaxis is caused by H+ ions rather than by the anions. A similar pheno-menon is known for Paramecium caudatum (Jennings, 1906), Polytoma uvella(Pringsheim and Mainx, 1926), Euglena gracilis (Mainx, 1928), and Astoriaocellata (ibid.).

To alkalis no clear reaction was observed. Protein-decomposition products,such as yeast-extract, beef-extract, bacto-tryptone, and bacto-peptone, havea strong attractive effect. The threshold for yeast-extract (dry, Difco) is at01 percent.; for bacto-peptone at 0-05 percent.; for the other two substancesat o-ooi per cent.; while o-1-0-2 per cent, of these causes very strong positivechemotaxis.

Starch grains do not exhibit an attractive action when presented in acapillary tube, while carmine, which is engulfed so plentifully, causes strongattraction in these conditions. Even very dilute suspensions of carmine incapillaries cause positive chemotaxis.

The impression that dead individuals of Euglena are sought out by Pera-nema with the help of a chemical sense was also tested by the capillary method.Euglena spirogyra or E. viridis, killed by moderate heating in CaSO4 solution,were sucked into capillaries so that they lay at a short distance from the openend, and the Peranema individuals in the watch-glass could not touch them.Many Peranetnae approached the opening of the capillary, some of themgliding forward until they came near to the dead Euglena. Even old culturefluid, freed from Euglena by centrifuging, caused positive reaction. As this


medium is slightly alkaline, the positive reaction cannot be caused by carbonicacid discharged by Euglena. The fresh, unused culture medium is notattractive.

In a similar way it was shown that living or heat-killed Saccharomycesextguus exude chemotactically attractive substances which can be washed outand tested with a positive result.

The mode of reaction can easily be watched, owing to the slowness ofmovement (Text-fig, n ) . It is clear that Peranema turns back when about to

leave a diffusion zone into which it has penetrated by chance, exhibiting phobo-chemotaxis. Whether the chemotaxis of Peranema could be described asklino-kinesis can only be decided by special investigations.

I wish to thank Dr. E. G. Pringsheim for his suggestion to use his cultureof Peranema for elucidating the biology of this organism, and for his adviceand encouragement during the two years and a half I worked at Cambridge.Thanks are also due to Dr. V. E. Coslett, Cavendish Laboratory, for his helpin electron microscopy, and to Dr. L. E. R. Picken, Department of Zoology,for his introduction into the use of protein dispersing agents for studyingthe flagellar structure.


REFERENCESBROWN, H. P., 1945. 'On the structure and mechanics of the protozoan flagellum.' Ohio

J. Sci., 45, 247.1946. 'On the structure and mechanics of the protozoan flagellum.' Abst. Dissert. The

Ohio State Univers., 49, 25.BROWN, V. E., 1930. 'The cytology and binary fission of Peranema.' Quart. J. micr. Sci., 73,

4°3-BCTSCHLI, O., 1876. 'Beitrage zur Kenntnis der Flagellaten, &c.' Zeit. wiss. Zool., 30, 205.

1883-7. 'Protozoa' in Bronn's Klassen und Ordnungen des Thierreichs. Bd. 1. Leipzigand Heidelberg (Winter).

BUTTERFIELD, C. T., 1929. 'Experimental studies of natural purification in polluted waters. III.A note on the relation between food concentration in liquid media and bacterial growth.'U.S. Publ. Health Repts., 44, 2872.

CHADEFAUD, M., 1938. 'Nouvelles recherches sur l'anatomie comparee des Eugleniens: lesPeranemines.' Rev. Algol., i r , 189.

DAWSON, J. A,, and BELKIN, M., 1928. 'The digestion of oils by Amoeba dubia.' Proc. Soc.exp. Biol. and Med., 36, 790.

1929. 'The digestion of oils by Amoeba proteus.' Biol. Bull., 56, 80.DEFLANDRE, G., 1934a. 'Sur la structure des flagelles.' Ann. Protist., 4, 31.

I934&- 'Existence sur les flagelles, de filaments lateraux ou terminaux (mastigonemes).'C. R. Acad. Sci., 198, 497.

DELLINGER, O. P., 1909. 'The cilium as a key to the structure of contractile protoplasm.'J. Morph., 20, 171.

DOFLEIN, F., 1916. Lehrbuch der Protozoenkunde. Jena (Fischer).FINLEY, H. E., 1930. 'Toleration of freshwater protozoa in increased salinity.' Ecology, 11,

337-GRAY, J., 1922. 'The mechanism of ciliary movement.' Proc. Roy. Soc. B, 93, 104.

1931- A textbook of experimental cytology. Cambridge (Univ. Press).HALL, R. P., 1926. 'Reactions of certain cytoplasmic inclusions to vital dyes and their relation

to mitochondria and Golgi apparatus in the flagellate Peranema trichophorum.' J. Morph.,48, 105.1933a. 'The method of ingestion in Peranema trichophorum and its bearing on the

pharyngial rod ('Staborgan') problem in Euglenida.' Arch. Protistenk., 81, 308.19336. 'The question of the ingestion of solid particles by Euglena.' Trans. Amer.

micr. Soc., 5a, 220.1934- 'A note on the flagellar apparatus of Peranema trichophorum and the status of the

family Peranemidae Stein.' Ibid., 53, 237.IQ37- 'Certain culture reactions of several species of Euglenidae.' Ibid., 56, 285.and POWELL, M. N., 1927. 'A note on the morphology and systematic position of the

flagellate Peranema trichophorum.' Ibid., 46, 155.ioa8. 'Morphology and binary fission of Peranema trichophorum. (Ehrenb.) Stein.'

Biol. Bull., 54, 36.HARDIN, G., 1942. 'An investigation of the physiological requirements of a pure culture of

the heterotrophic flagellate, Oikomonas termo Kent.' Physiol. Zool., 15, 466.1944. 'Physiological observations and their ecological significance. A study of the

protozoan, Oikomonas termo.' Ecology, 25, 192.HOLLANDE, A., 1942. 'Etude cytologique et biologique de quelques Flagelles libres." Arch.

Zool. exp. gen., 83, 1.HYMAN, L. H., 1936. 'Observations on Protozoa: I. The impermanence of the contractile

vacuole in Amoeba vespertilio. II. Structure and mode of food ingestion in Peranema.'Quart. J. micr. Sci., 79, 43.

JAHN, T. L., 1931. 'Studies on the physiology of the euglenoid flagellates. III. The effect ofhydrogen ion concentration on the growth of Euglena gracilis Klebs.' Biol. Bull., 61, 387.

JAMES-CLARK, H., 1868. 'On the spongiae ciliatae as infusoria flagellata.' Ann. Mag. Nat.Hist., i, 250.

JENNINGS, H. S., 1906. The behavior of the lower organisms. New York (Columbia UniversityPress).

JOLLOS, V., 1925. 'Flagellata.' Handbuch der Zoologie, pp. 115 sqq. Berlin und Leipzig(Walter de Gruyter).


KENT, W. S., 1880-2. A manual of the infusoria. London (Bogue).KLEBS, G., 1883. 'l)ber die Organisation einiger Flagellatengruppen, &c.' Unters. Botan.

Inst. Tubingen, i, Z33.1893. 'Flagellaten-Studien.' Zeit. wiss. Zool., 55, 265.

KORSHIKOFF, A., 1923. 'Ober den Bau und die Aggregation der Geisseln bei den Volvocalesund den Flagellaten.' Arch. russ. Protist., 2, 195. (German summary.)

KRIJGSMAN, B. J., 1925. 'Beitrage zum Problem der Geisselbewegung.' Arch. Protistenk.,52, 478-

LACKEY, J. B., 1929. 'Studies in the life-histories of Euglenida. II. The life-cycles of Ento-siphon sulcatum and Peranema trichophorum.' Arch. Protistenk., 67, 128.1933- 'Studies in the life-histories of Euglenida. III. The morphology of Peranema

trichophorum.' Biol. Bull., 65, 238.LEMMERMANN, E., 1913. 'Euglenineae' in Die Siisstvasserflora Deutschlands, Osterreichs, etc.

Jena (G. Fischer).LILLY, D. M., 1942. 'Nutritional and supplementary factors in the growth of carnivorous

ciliates.' Physiol. Zool., 15, 146.LOEFER, J. B., 1931. 'Morphology and binary fission of Heteronema acus (Ehrbg.) Stein.'

Arch. Protistenk, 74, 449.LOWNDES, A. G., 1936. 'Flagellar movement.' Nature, 138, 210.

1941- 'On flagellar movement in unicellular organisms.' Proc. zool. Soc. Lond., 111, i n .1944a. 'The swimming of unicellular flagellate organisms.' Ibid., 113, 99.1944&. 'The swimming of Monas stigmatica Pringsheim and Peranema trichophorum

(Ehrbg.) Stein and Volvox sp. Additional experiments on the working of a flagellum.'Ibid., 114, 325.

LUND, E. J., 1914. 'The relations of Bursaria to food. II. Digestion and resorption in the foodvacuole and further analysis of the process of extrusion.' J. exp. Zool., 17, 1.

MAINX, F., 1928. 'Beitrage zur Morphologie und Physiologie der Eugleninen, I, II.' Arch.Protistenk, 60, 305, 355.

MASSART, J., 1920. 'Recherches sur les organismes inferieurs VIII. Sur la motilite des Flagel-lates.' Bull. Cl. Sci. Acad. Royale Belgique, seance d'avril 1920, 116.

MAST, S. O., 1912. 'The reactions of the flagellate Peranema.' J. Anim. Behav., 2, 91.1938. 'Digestion of fat in Amoeba proteus.' Biol. Bull., 75, 389.1942. 'The hydrogen-ion concentration of the content of the food vacuoles and the

cytoplasm in Amoeba and other phenomena concerning the food vacuoles.' Ibid., 83, 173.and BOWEN, W. J., 1944. 'The food-vacuole in the Peritricha, with special reference to

the hydrogen-ion concentration of its content and of the cytoplasm.' Ibid., 87, 188.and HAWK, B., 1936. 'Response to light in Peranema trichophorum.' Ibid., 70, 408.

PFEFFER, W., 1884. 'Lokomotorische Richtungsbewegungen durch chemische Reize.' Unters.bot. Inst. Tubingen, 1, 363.1888. 'Uber chemotactische Bewegungen von Bakterien, Flagellaten und Volvocineen.'

Ibid., 2, 582.PITELKA, D. R., 1945. 'Morphology and taxonomy of flagellates of the genus Peranema

Dujardin.' J. Morph., 76, 179.PRINGSHEIM, E. G., 1928. 'Physiologische Untersuchungen an Paramaecium Bursaria.' Arch.

Protistenk., 64, 289.1948. 'Taxonomic problems in the Euglenineae.' Biol. Rev., 23, 46.and HOVASSE, R., 1950. 'Les relations de parente entre Eugl£nacees et Astasiacees.'

Arch. Zool. exp. gen., 86, 499.and MAINX, F., 1926. 'Untersuchungen an Polytoma uvella Ehrbg., insbesondere uberBeziehungen zwischen chemotaktischer Reizwirkung und chemischer Konstitution.'Planta, 1, 583.

RHODES, R. C, 1926. 'Mouth and feeding habits of Heteronema acus (The mouth of thePeranemidae Klebs, involving the classification of the Euglenoidina relative to the newfamily Heteronemidae Calkins).' Anat. Rec, 34, 152.

SCHAEFFER, A. A., 1918. 'A new and remarkable diatom-eating flagellate, Jenningsia dialo-mophaga.' Trans. Amer. micr. Soc, 37, 177.

SCHMITT, F. O., HALL, C. E., and JAKUS, M. A., 1943. 'The ultrastructure of protoplasmicfibrils.' Biol. Symposia, 10, 261.

SHAPIRO, N. N., 1927. 'The cycle of hydrogen-ion concentration in the food-vacuoles ofParamaecium, Vorticella, and Stylonychia.' Trans. Amer. micr. Soc, 46, 45.


SHETTLES, L. B., 1937. 'Response to light in Peranema trichophorum with special reference todark adaptation and light adaptation.' J. exp. Zool., 77, 215,

SHIPLEY, P. G., and DE GARIS, C. G., 1925. 'The third stage of digestion in Paramaecia.'Science, 62, 266.

SHORTESS, G. S., 1942. 'The relation between temperature, light and rate of locomotion inPeranema trichophorum in response to changes in temperature.' Physiol. Zool., 15, 184.

SKUJA, H., 1948- 'Taxonomie des Phytoplanktons einiger Seen in Uppland, Schweden.'Symb. Bot. Uppsaliens., 9, 3.

STEIN, F. R. V., 1878. Der Organismus der Infusionsthiere. Abt. III. Der Organismus derFlagellaten, Halfte I. Leipzig (Engelmann).

TANNREUTHER, G. W., 1923. 'Nutrition and Reproduction in Euglena.' Arch. Entwick.Mech. 52, 367.

VLK, W., 1938. 'Ober den Bau der Geissel.' Arch. Protistenk., 90, 448WILBER, C. G., 1942. 'Digestion of fat in the rhizopod, Pelomyxa carolinensis.' Biol. Bull, 83,

320.

investigations of the biology of peranema trichophorum ...anterior end and a tapering posterior end...

Documents